Biology LVI - microscopy

magnification

the number of times larger an image appears compared to the size of the object viewed

resolution

the shortest distance between 2 objects that are still seen as separate objects. the higher the resolution the greater the detail.

how do light microscopes work

visible light passes through specimen and is then mangified

light - resolution

limited (low) objects closer than 200nm are 1 object

light - magnification

up to 2000x, school = 400x

preparations/conditions for light microscopy

thin sections cut onto slide + staining necessary

specimen for light microscopy

can be live, blood smears, sections of cell tissue

pros of light microscopy

cheap, easy to use, portable, can study whole organism if dissection microscope is used

cons of light microscopy

low resolution, low magnification (cannot clearly see cell ultrastructure)

2 lenses in light microscopy

eyepiece + objective

wavelength of light used for light microscopy

400-700nm

benefits of staining

makes structures more visible, increase contrast (cells are transparent due to water), more cells + structures visible, identification of organelles, cell types, tissues, specific molecules

issues with staining

• may require long protocols

• sometimes specific conditions are needed for staining to work

how does a transmission electron microscope (TEM) work

uses a beam of electrons which are controlled by condenser magnet, thee pass through objective and projector lens onto a screen. Different thicknesses within sample create contrast.

TEM resolution

high - 0.05-1nm

TEM magnification

over 1,000,000x

TEM wavelengths used

0.004nm

preparations/conditions for TEM

• dead material only

• done in vacuum

• stained with metal salts + dehydrated

TEM pros

can see detailed ultrastructure, superior magnification and resolution

cons of TEM

• preparation can cause artefacts

• expensive

• training required

added TEM detail

very thin sample so electrons can penetrate

scanning electron microscope (SEM) how does it work

beam of electrons transmitted across surface of gold/pallodium coated specimen- detects secondary electrons

TEM image formed

2D black and white

SEM image formed 

3D black and white (can add false colour)

SEM resolution

0.4-20nm

SEM magnification

500,000x

wavelengths used SEM

0.004nm

SEM preparations/conditions

gold/pallodium coating of specimen, dead sample.

pros of SEM

• 3D at high resolution

• superior magnification

cons of SEM

• very expensive

• metallic film may be toxic

• training required

magnification equation

image size/ actual size (must be same units)

mm

x10^-3

micro metre (mew m)

x10^-6

nm

x10^-9

cm

x10^-2

iodine in potassium iodide stains….

cellulose yellow + starch granules blue/black

acetic orcein stain

stains DNA dark red so chromosomes can be seen 

eosin stain

stains cytoplasm

sudan red stains

lipids

methylene blue

acidic animal cell components

eye piece graticules are used for

to measure the actual size of a specimen- they’re etched onto the eyepiece- each division will give you a different value depending on what magnification is used.

to calculate the true length of EPU (eyepiece unit)

align the eyepiece graticule with a stage micrometre

length of EPU =

total length of eyepiece graticule/number of eyepiece divisions

why do you need to use the same eyepiece lens when measure EPU

so that eyepiece graticule remains the same therefore length of eyepiece unit will remain the same

metabolism

the building up and breaking down of molecules

cytoplasm

made of cytosol - which is made up of water, salts and organic molecules

nucleus description

largest organelle, membrane bound, chromatin (form when chromosomes aren’t dividing)

nucleus function

contain genetic info in form of DNA, DNA controls the metabolic activity of the cell + many of the proteins/enzymes necessary

describe nucleolus 

darkly staining area within the nucleus

nucleolus function

responsible for synthesis of ribosomal RNA and formation of ribosomes

nuclear envelope

double membrane + has pores

nuclear envelope fucntion

protect nucleus from damage (cytoplasm) + allow molecules to move into and out of the nucleus

rough endoplasmic reticulum 

flattened sacks called cisternae (fluid filled), continuous with nuclear envelope, ribosomes on them 

RER function

responsible for the synthesis and transport of proteins (vesicles)

smooth endoplasmic reticulum

flattened sacks called cisternae, fluid filled, no ribosomes

SER function

lipid + carb synthesis + storage

golgi apparatus

cisternae - flattened membrane sacs

golgi function

modify proteins + packing them into vesicles

ribosomes

very small - proteins and RNA made from 2 subunits

ribosome function

site of protein synthesis

mitochondria

double membrane (folded inner membrane) = cristae, fluid inside called matrix which contained enzymes, DNA, Ribosomes

mitochondria function

site of final stage of cellular respiration - synthesis of ATP

lysosomes

membrane bound sac, enzymes and proteins held in its membrane 

lysosome function

breakdown of waste in cells (old organelles), break down of pathogens ingested by phagocytes

plasma membrane

phospholipid bilayer, proteins embedded in membrane

centrioles

small tubes of microtubules near nucleus

centriole function

2 associated centrioles= centrosome = assembly and organisation of spindle fibres in cell division

flagella use=

propulsion

cilia use

moving substances over cell

chloroplasts=

large, double membrane, grana = stacks of thylakoids filled with chlorophyll, surrounding this is fluid called stroma containing enzymes + DNA

chloroplast function

site of photosynthesis

cell wall=

cellulose lattice, embedded in calcium pectate (pectin) (glue), holes in walls linking cells = plasmodesmata

cell wall function

give shape, rigidity, defence mechanism for pathogens, structure

vacuole

filled with sap (fluid, minerals, sugar) surrounded by tonoplast (membrane)

vacuole function

maintenance of turgor (rigidity), tonoplast = selectively permeable, contains sap

cytoskeleton

the complex network of proteins present within the cytoplasm

function of cytoskeleton

• provide mechanical strength

• movement within cell

• movement of cell itself

molecules that make up cytoskeleton

• microfilaments

• intermediate filaments

• microtubules

• motor proteins

microfilaments

made from protein actin, 7nm in diameter

intermediate filaments

range of proteins, can extend between cells + stabilise tissues

microtubules

made from protein tubulin (18-30nm diameter), they form spindle + make up undulopia

motor proteins (dyneins)

act as molecular motors, have active site to hydrolyse ATP

microfilament function

• maintain shape

• provide mechanical shape

• allow cell to move

intermediate filament function

surround nucleus and hold it in place, allow cells to stick together + communicate

microtubules function

provide shape, support and tracks for movement of vesicles/organelles. Motor proteins walk along track pulling organelle

eukaryotic flagella (protoctist)

9 microtubule doublet + 2 central microtubules next to dynein arms, these change shape causing microtubule to slide moving whole axoneme in a whip-like motion

prokaryotic flagella

rotating disk spins spiral protein using ATP

how is cytoskeleton involved in mitosis

spindle attached to the centrioles pulls chromosomes to opposite ends of the cell

centrioles extra details

organising centres + only animals

division of labour

specialised functions of cell organelles that work together to ensure the cells survival and performance.

secretion of extracellular proteins MA

• nucleus = transcription, DNA copied to mRNA which leaves nucleus via nuclear pores

• RER (ribosomes) = translation. Codon sequence on mRNA is used to create a polypeptide

• polypeptide → RER lumen + chaperone proteins fold polypeptide

• Protein leaves RER to a transport vesicle, travels along microtubes using motor proteins to the golgi

• golgi= post translation modification of protein as proteins moves through cisternae of golgi (eg formation of glycoproteins)

• proteins packaged into secretory vesicles and transported to plasma membrane where they fuse + release their contents outside of the cell (exocytosis)

prokaryote to eukaryote size

P =0.2-2micrometre E=10-100micrometre

Prokaryotes

simple structure, no membrane bound organelles, smaller ribosomes (70s where E=80s), 100-1000 times smaller than eukaryotic

s

svedbergs (density)

organelles of eukaryotic cells used to be

prokaryotic cells before endosymbiosis eg: mitochondria + chloroplast

endosymbiosis 

2 organisms living together with one living inside the another benefitting both parties

features of a prokaryotic cell

• nucleoid (naked/free DNA)

• Mesosome (respiration)

• Peptidoglycan cell wall

• capsule

• smaller ribosomes

• Plasmids

• Pilli (attachment to other bacterial cells + exchange of plasmids)

• Plasma membrane

• flagella with rotating base

how do prokaryotes divide

binary fission

Mesosome

where ATP is synthesised in prokaryote

flagella in prokaryotes

made from spiral protein flagellin